Purification and characterisation of a quorum quenching AHL-lactonase from the endophytic bacterium Enterobacter sp. CS66.

The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent.

Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 µM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 µM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 µM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.

Isolation and characterization of xanthan-degrading Enterobacter sp. nov. LB37 for reducing the viscosity of xanthan in petroleum industry.

A Gram-negative, straight rod and facultative anaerobic bacterium was isolated from soil sample. It exhibits the phenotypic characteristics consistent with its classification in the genus Enterobacter. The isolate ferment glucose to acid and gas. Arginine dihydrolase, ornithin decarboxylase and gelatinase but not deoxyribonuclease was produced by this isolate. There was no hydrogen sulfide production. On the basis of the phenotypic data, together with phylogenetic analysis based on 16S rDNA gene sequences, this strain should represent a novel species of the genus Enterobacter and was designated as LB37. The strain LB37 could degrade xanthan molecules resulting in the rapid decrease of the viscosity of xanthan solution used in oil drilling process. Endoxanthanase activity was also detected in the culture supernatant. To our knowledge, it is the first report on the microbes being involved in the xanthan degradation for oil industry. The isolate LB37 would be useful for potential application in enhanced oil recovery and oil drilling field.

Evaluation of a new cefepime-clavulanate ESBL Etest to detect extended-spectrum beta-lactamases in an Enterobacteriaceae strain collection.

OBJECTIVES: In this study, we evaluated the performance of a new ESBL Etest configuration based on clavulanate synergy with cefepime compared with cefotaxime-clavulanate and ceftazidime-clavulanate ESBL Etest strips for the detection of extended-spectrum beta-lactamases (ESBL) in an Enterobacteriaceae strain collection, with special focus on Enterobacter spp. METHODS: Overall, a total of 54 clinical isolates of ESBL-producing Enterobacteriaceae species were evaluated: Enterobacter aerogenes (n=3), Enterobacter cloacae (n=10), Escherichia coli (n=10), Klebsiella oxytoca (n=3), Klebsiella pneumoniae (n=25) and Proteus mirabilis (n=3). To check Etest behaviour with resistance phenotypes similar to ESBL, our panel was expanded by six clinical isolates of K. oxytoca that were identified as putative producers of their chromosomal K1 beta-lactamase. RESULTS: With this panel, ESBL Etest was 98% sensitive with cefepime-clavulanate, 83% with cefotaxime-clavulanate, and 74% with ceftazidime-clavulanate strips. Concentrating on Enterobacter spp., reliable ESBL detection could only be achieved by the new cefepime-clavulanate strip since it confirmed ESBL production in all strains (100% sensitivity) whereas only 4/13 (31%) of Enterobacter strains were positive using cefotaxime-clavulanate or ceftazidime-clavulanate strips. A limitation of using the new cefepime strip was less than optimal specificity with K1 phenotypes of K. oxytoca: among six strains, four isolates were scored false-positive by Etest strips containing cefepime-clavulanate. CONCLUSION: The new Etest ESBL strip containing cefepime-clavulanate is a valuable supplement to current methods for detection of ESBLs. In our study collection, the cefepime-clavulanate strip was the best configuration for detection of ESBLs, particularly in Enterobacter spp.

Treatment of a meningitis due to an Enterobacter aerogenes producing a derepressed cephalosporinase and a Klebsiella pneumoniae producing an extended-spectrum beta-lactamase.

A case of nosocomial meningitis due to a Klebsiella pneumoniae producing a CAZ-5 extended-spectrum beta-lactamase and an Enterobacter aerogenes producing a derepressed cephalosporinase is reported. The intrathecal catheter incriminated was removed and a treatment with ceftazidime (4 g/24 h) and amikacin (1.5 g/24 h) was started. After 24 h ceftazidime was replaced by imipenem (2 then 4 g/24 h). This treatment failed to obtain cerebrospinal fluid sterilization; therefore the imipenem dosage was increased to 8 g/24 h and two intrathecal infusions of amikacin (50 mg) were carried out. Thereafter the patient recovered.

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae.

Several species of Enterobacteriaceae were investigated for their ability to synthesize selenium-containing macromolecules. Seleniated tRNA species as well as seleniated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobically induced formate dehydrogenase isoenzymes of E. coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunological and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hybridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of conservation of the seleniating system throughout the enterobacteria.

L-2,4-diaminobutyric acid decarboxylase activity responsible for the formation of 1,3-diaminopropane in Enterobacter aerogenes.

High content of 1,3-diaminopropane (DAP), a normally minor derivative of polyamine metabolism, have been observed in cells of Enterobacter aerogenes. Supplementation of the growth medium with L-2,4-diaminobutyric acid (L-DABA) resulted in increased production of DAP, but not if supplemented with spermidine. On the basis of these observations, the biosynthetic route for DAP was evaluated. It has appeared that this bacterium possesses a novel enzyme activity catalysing the formation of DAP from L-DABA. Lack of the activity for oxidative cleavage of spermidine yielding DAP suggests that the enzyme termed DABA decarboxylase is responsible for the formation of DAP in this bacterium. The enzyme was partially purified 360-fold and some properties were examined. The pH optimum for the activity was 7.75-8.0, and the enzyme showed an absolute requirement for pyridoxal 5'-phosphate with the Km value of 41 microM. The Km value for L-DABA was 0.32 mM, and neither L-2,3-diaminopropionic acid, L-ornithine nor L-lysine showed detectable substrate activity towards the partially purified enzyme. Mg2+ and dithiothreitol greatly activated the enzyme.

Resistance emerging after pefloxacin therapy of experimental Enterobacter cloacae peritonitis.

Resistance emerging after pefloxacin therapy was investigated in an experimental Enterobacter cloacae infection. Mice were inoculated intraperitoneally (mean inoculum, 0.9 X 10(8) CFU) with one of four strains initially susceptible to quinolones and treated with a single 25-mg/kg dose of pefloxacin. This therapy produced a net decrease of bacterial counts in the peritoneal fluid, but with the of the isolates, posttherapy (PT1) strains emerged with decreased susceptibilities to quinolones (4- to 1,024-fold), to the structurally unrelated antibiotics (4- to 16-fold) chloramphenicol and trimethoprim, and sometimes to tetracycline and beta-lactam compounds. In a second set of experiments, new mice were similarly infected with PT1 strains and treated with up to five 25-mg/kg doses of pefloxacin. Compared with parent isolates, PT1 strains produced similar disease and peritoneal bacterial count in the control animals. In treated mice posttherapy (PT2) strains emerged that showed 8- to 64-fold increases in quinolone MICs compared with the PT1 strains inoculated. All PT1 and PT2 strains showed altered outer membrane protein patterns, principally marked by a decreased 37,000-molecular-weight band generally accompanied by an increased 42,000-molecular-weight band. Whole cells from all PT1 and PT2 strains, exposed to [14C]pefloxacin for 15 to 60 s, bound significantly less radioactivity than the corresponding parent strains. After partial purification, DNA gyrase extracted from the most resistant isolates (one PT1 and the PT2 strains) showed a 100- to 450-fold 50% inhibitory concentration increase for pefloxacin. Altogether, pefloxacin can select in vivo two types of resistant strain, one with only decreased permeability and another with decreased permeability combined with altered DNA gyrase.

Relative substrate affinity index values: a method for identification of beta-lactamase enzymes and prediction of successful beta-lactam therapy.

Using a nitrocefin competition assay, I determined the relative substrate affinity index (RSAI) values of nine clinically significant beta-lactamase enzymes against a range of beta-lactams. Using selected beta-lactam substrates, I observed large differences in the RSAI values of the nine enzymes that were sufficient in many cases to positively identify specific enzymes. I made use of the unique RSAI values of SHV-1, TEM-1, and TEM-2 beta lactamases with cefoxitin to screen for the presence of these enzymes in Klebsiella aerogenes clinical isolates. The RSAI values also allow for the prediction of the outcome of beta-lactam therapy against specific beta-lactamase-producing isolates.

Broad-spectrum beta-lactam resistance in Enterobacter: emergence during treatment and mechanisms of resistance.

Of three patients with Enterobacter mediastinitis treated with cefamandole, two failed initial treatment due to emergence of strains resistant to cefamandole and third generation cephalosporins. Extracts of the sensitive strains showed inducible hydrolysis of cephalothin; resistant strains (wild and laboratory derived) showed constitutive hydrolysis of cephalothin and cefamandole and bound but did not hydrolyze cefotaxime and latamoxef (moxalactam). In one mutant, increased latamoxef resistance appeared due to non-enzymatic mechanism(s). The Enterobacter strain from the patient who was treated successfully with cefamandole yielded only low level resistant mutants with lower cephalosporin binding than obtained with the other strains.

Induction of adenosine deaminase in Escherichia coli.

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.